Etiologic Risk Group (see link below):
N/A */Disease Information (sort of like the Intro to your Mini Research Write up):
Tuberculosis is a contagious bacterial infection is spread through the air. Tuberculosis usually attacks the lungs and is caused by a bacteria called Mycobacterium tuberculosis. The symptoms of Tuberculosis include cough, coughing up blood, chest pain and fatigue. If the Tuberculosis infection worsens or goes untreated, it can infect the central nervous system, kidneys, lungs, heart and brain.
Link to TDR Targets page (if present):
N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) https://www.ncbi.nlm.nih.gov/gene/887070 Essentiality of this protein:
The only predicted protein phosphatase in M.tuberculosis, it dephosphorylates at least 5 protein kinases (PknA, PknB, PknD, PknE and PknF) and the penicillin-binding protein PBPA - Dr. B 060617 Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Ortholog of A. thaliana: monomer
Enzyme Assay information (spectrophotometric, coupled assay ? The purified wild-type or mutant proteins were exchanged into reaction buffer containing 50 mM NaCl, 20 mM Tris, pH 7.5, and 0.5 mM TCEP. The enzymes were incubated with various amounts of pNPP, and reaction progress data were collected at 405 nM and 25°C using a SPECTRAmax 190 spectrophotometer (Molecular Devices). Kinetic constants were calculated using SigmaPlot (Systat Software Inc.). In order to determine the effect of metal concentration on enzyme activity, various amounts of MnCl2 were mixed with 50 mM pNPP. The reaction was initiated by adding enzyme at a final concentration of 1 μM, and the assay was monitored continuously for several minutes. To determine the kcat and Km values for pNPP, eight substrate dilutions were made. Reactions contained 100 mM MnCl2 and a final enzyme concentration of 1 μM.
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: ---- Max % Identities: ---- % Positives ---- Chain used for homology:
Current Inhibitors: Phosphoserine, phosphothreonine, para-nitrophenol phosphate Expression Information (has it been expressed in bacterial cells): Purification Method: recombinant wild-type and selenomethionine-labeled enzymes from Escherichia coli strain BL21(DE3) by gel filtration and ion exchange chromatography Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
<span style="font-family: monospace,serif;">>AAF04553.1 putative protein phosphatase type 2C, partial [Caenorhabditis brenneri]
<span class="ff_line">SKVSQYSGINLHKKVVARKEFSEGNLKGAIERGFLDLDQQMRIDEETKDDVSGTTAVVVLIKEGDVYCGN
AGDSRAVSSVVGEARPLSFDHKPSHENEARRIIAAGGWVEFNRVNGNLALSRALGDFAFKNCDTKPAEEQ
IVTAYPDVITDKLTPDHEFIVL</span></span>
*length of your protein in Amino Acids
162 aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 17762.90 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 10095 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*GC% Content for gene: 67.572816 *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): Forward Primer: TACTTCCAATCCATGACCCTCGTTCTGCGTTATGC
*NCBI Gene # or RefSeq#: 887070
*Protein ID (NP or XP #) or Wolbachia#: AF177866_1
*Organism (including strain):
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Etiologic Risk Group (see link below):
N/A
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Tuberculosis is a contagious bacterial infection is spread through the air. Tuberculosis usually attacks the lungs and is caused by a bacteria called Mycobacterium tuberculosis. The symptoms of Tuberculosis include cough, coughing up blood, chest pain and fatigue. If the Tuberculosis infection worsens or goes untreated, it can infect the central nervous system, kidneys, lungs, heart and brain.
Link to TDR Targets page (if present):
N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.ncbi.nlm.nih.gov/gene/887070
Essentiality of this protein:
The only predicted protein phosphatase in M.tuberculosis, it dephosphorylates at least 5 protein kinases (PknA, PknB, PknD, PknE and PknF) and the penicillin-binding protein PBPA - Dr. B 060617
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Ortholog of A. thaliana: monomer
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://www.ebi.ac.uk/enzymeportal/search/P9WHW5/enzyme
Link to PDB:
http://www.rcsb.org/pdb/explore.do?structureId=1TXO
*EC#: 3.1.3.16
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.16&Suchword=&reference=&UniProtAcc=&organism%5B%5D=Mycobacterium+tuberculosis&show_tm=0
Enzyme Assay information (spectrophotometric, coupled assay ?
The purified wild-type or mutant proteins were exchanged into reaction buffer containing 50 mM NaCl, 20 mM Tris, pH 7.5, and 0.5 mM TCEP. The enzymes were incubated with various amounts of pNPP, and reaction progress data were collected at 405 nM and 25°C using a SPECTRAmax 190 spectrophotometer (Molecular Devices). Kinetic constants were calculated using SigmaPlot (Systat Software Inc.).
In order to determine the effect of metal concentration on enzyme activity, various amounts of MnCl2 were mixed with 50 mM pNPP. The reaction was initiated by adding enzyme at a final concentration of 1 μM, and the assay was monitored continuously for several minutes. To determine the kcat and Km values for pNPP, eight substrate dilutions were made. Reactions contained 100 mM MnCl2 and a final enzyme concentration of 1 μM.
http://www.sciencedirect.com/science/article/pii/S096921260400334X
http://www.sciencedirect.com/science/article/pii/S0006291X03020011?via%3Dihub
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
http://www.uvm.edu/~bio1and2/lab/Lab%20manuals%20Fall%202011/Enzyme%20Activity%20Phosphatase.pdf
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: Phosphoserine, phosphothreonine, para-nitrophenol phosphate
Expression Information (has it been expressed in bacterial cells):
Purification Method:
recombinant wild-type and selenomethionine-labeled enzymes from Escherichia coli strain BL21(DE3) by gel filtration and ion exchange chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids
162 aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 17762.90 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 10095
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as textonly):
*GC% Content for gene:
67.572816
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer:
TACTTCCAATCCATGACCCTCGTTCTGCGTTATGC
Reverse Primer:
TATCCACCTTTACTGTTAGTGTTCGAGGTCCGCAA**